Abstract This direct SBIR Phase II project aims to develop a novel technology and associated assays for efficiently and robustly extracting circulating miRNAs from blood (i.e. plasma or serum), which could transform the utility of miRNA testing in the diagnosis and monitoring of major diseases such as cancer and cardiovascular disease. Our competitive advantage lies in the ability to retrieve circulating miRNAs more efficiently and robustly than current extraction methods, which has been a critical barrier in implementing miRNA testing clinically. This advantage is achieved by implementing sequence specific capture in conjunction with a propriety method of making novel capture beads. Circulating miRNAs are potential disease biomarkers. However, to date, there are no FDA approved miRNA tests available. A bottleneck problem is unreliable circulating miRNA extraction. Current extraction methods are based on adsorption of polar molecules on polar surfaces, which were originally developed to extract large DNA and RNA molecules. However, because miRNAs are so small (~22nt), their interaction with polar surfaces is much weaker, therefore their adsorption on polar surfaces can be easily interrupted by other molecules present in sample. Since the weak adsorption of miRNAs on polar surfaces is an inherent problem that cannot be fully solved even if all other conditions were optimized, new methods for retrieving circulating miRNAs based on a different mechanism are clearly needed. Sequence-specific capture (SSC) is another method to extract nucleic acids, but historically it performs poorly when used to extract circulating nucleic acids from clinical samples due to the lack of effective capture beads. In addition, the cost of making capture beads by current methods is very high, making SSC too expensive for clinical use. Recently, we made a major breakthrough by developing a proprietary method of making capture beads that can transform SSC from a concept to a practical method for extracting circulating miRNAs in an efficient, robust, and cost-effective manner. Therefore, we employed our capture beads to develop our own SSC assays, and conducted a systematic study to examine the feasibility of using our SSC assay in extracting circulating miRNAs. Our SSC assay was indeed found to efficiently and robustly extract circulating miRNAs. In the study, we also found that extraction efficiency of current kits varied by as much as 60-fold by plasma sample, further confirming that current methods are not robust. Considering the problem of current methods, unparalleled features of our SSC technology, and success of our early study, we propose this Phase II SBIR project to further develop and validate our SSC technology for extracting circulating miRNAs from plasma/serum, providing a foundation for its commercialization.